Targeting brown adipose tissue content or activity has therapeutic potential for treating obesity, diabetes and the metabolic syndrome by increasing energy expenditure. Understanding of the differentiation and function of human adipose tissue is key to formulating therapeutic interventions to treat these metabolic disorders. A major impediment for these studies is the lack of human-derived brown, beige and white fat progenitor cell models. We have successfully generated pairs of immortalized human brown and white fat precursor cells from human neck adipose tissue and characterized their adipogenic differentiation and metabolic function. Using clonal analysis and gene expression profiling, we have defined novel genetic biomarkers in human preadipocytes that could predict the thermogenic potential of mature adipocytes. In addition, using quantitative lipidomics analyses, we have identified new lipid mediators that are able to modulate brown fat-mediated thermogenesis. Building on these preliminary data, we hypothesize that brown fat-mediated thermogenic program is tightly regulated by both genetic and humoral factors. In this grant, we will directly test this hypothesis by determining the role of the identified genetic markers and liid mediators in adipocyte differentiation and mitochondrial functions using the paired human brown and white precursor cells. A series of gain- and loss-of-function experiments will be performed to thoroughly evaluate the impact of these factors on cellular metabolism. The in vivo effects of the human cells will be evaluated after transplantation into immune-deficient nude mice. We will also determine the molecular mechanisms that mediate the effect of these factors on modulating fuel utilization and mitochondrial function of brown or beige adipocytes. Finally, to explore the therapeutic potential of human brown and beige fat, we will test the utility of developing novel human cell surface markers for isolation, imaging and target delivery, and perform chemical screens using the human adipose cells carrying a brown fat specific reporter to identify potential drug candidates. Accomplishing the proposed studies will not only improve our current understanding of human adipocyte biology, but also open up new avenues for developing effective therapies for obesity, diabetes and other metabolic disorders.